( ...)Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

Fig. 3 A. Diagram of the isoamylase cDNA clone. The PCR-amplified 5' end consists of an overlapping sequence and an additional 5' area which completes the open reading frame. B. At the 5' end, the GC content of the isoamylase sequence is up to 80%, whereas the average GC content of the overall sequence is merely 52%.

With the aid of a simple PCR reaction, it was possible to amplify the 5' end of a cDNA coding for isoamylase directly from a ?ZAPII library. Conventional plaque screening of the same cDNA library produced several short isoamylase cDNAs, but no clone with a complete coding region. This finding may be attributable to the extremely high GC content of the 5' region of the isoamylase cDNA (Fig. 3b). In regions with high GC content, RNA is able to form stable secondary structures, which abort the cDNA synthesis and lead to shorter cDNA clones. Similarly, single-stranded DNA templates in a PCR reaction can form secondary structures that may interfere with amplification [3,4]. The higher the annealing temperature selected for the PCR reaction, the more likely secondary structures will be suppressed. This may explain why annealing temperatures of 69C or even 72C lead to more specific product than a temperature of 63C, although 63C is much closer to the theoretically optimal temperature of 61C. Determining a suitable stringent annealing temperature is crucial for obtaining specific products in the PCR. In this case, temperature gradients such as those on Mastercycler gradient enable systematic, time-saving experiments.